Mechanism of inhibition of Rho-dependent transcription termination by bacteriophage P4 protein Psu.
Mechanism of inhibition of Rho-dependent transcription termination by bacteriophage P4 protein Psu.
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DOI：
10.1074/jbc.m603982200
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发表时间：
2006-09-08
期刊：
影响因子：
--
通讯作者：
Sen R
中科院分区：
文献类型：
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作者：
Pani B;Banerjee S;Chalissery J;Muralimohan A;Loganathan RM;Suganthan RB;Sen R
来自噬菌体P4的外壳蛋白Psu已被证明在体内抑制Rho依赖性转录终止。Psu和Rho的共过表达导致细胞活力的丧失，这是蛋白质的抗Rho活性的结果。Psu的抗终止特性通过从其C末端缺失10或20个氨基酸或通过Rho中的突变Y80 C而被废除。所有这些实验都表明Rho和Psu之间可能存在相互作用。纯化的Psu蛋白本质上是α-螺旋的，并且似乎是二聚体。Rho和野生型Psu在亲和基质上的共纯化和它们两者在Superose-6凝胶过滤中的共洗脱表明这些蛋白质的直接缔合，而Psu的C末端10个氨基酸缺失衍生物在该测定中未能被拉下。这表明这些突变体的功能丧失与它们不能相互作用有关。体外终止实验表明Psu能以浓度依赖性方式特异性抑制Rho依赖性终止。Psu的存在影响了ATP的亲和力，降低了Rho的ATP酶活性，但不影响初级或次级RNA结合活性。在Psu的存在下，还观察到Rho从停滞的延伸复合物非常缓慢地释放RNA。我们认为Psu抑制Rho依赖性终止是通过减慢Rho沿着RNA的转位，因为它的ATP酶活性慢。
Psu, a coat protein from bacteriophage P4, has been shown to inhibit Rho-dependent transcription termination in vivo. Co-overexpression of Psu and Rho led to the loss of viability of the cells, which is the consequence of the anti-Rho activity of the protein. The antitermination property of Psu is abolished either by the deletion of 10 or 20 amino acids from its C terminus or by a mutation, Y80C, in Rho. All these experiments indicated probable interactions between Rho and Psu. Purified Psu protein is α-helical in nature and appeared to be a dimer. Co-purification of Rho and wild-type Psu on an affinity matrix and co-elution of both of them in Superose-6 gel filtration suggests a direct association of these proteins, whereas a C terminus 10-amino acid deletion derivative of Psu failed to be pulled down in this assay. This indicates that the loss of the function of these mutants is correlated with their inability to interact with each other. In vitro termination assays revealed that Psu can inhibit Rho-dependent termination specifically in a concentration-dependent manner. The presence of Psu affected the affinity of ATP and reduced the rate of ATPase activity of Rho but did not affect either primary or secondary RNA binding activities. In the presence of Psu, Rho was also observed to release RNA very slowly from a stalled elongation complex. We propose that Psu inhibits Rho-dependent termination by slowing down the translocation of Rho along the RNA because of its slow ATPase activity.